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0.2 μm nitrocellulose membrane

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The 0.2 μm nitrocellulose membrane is a laboratory filtration product designed for the separation and retention of small particles and molecules. It has a pore size of 0.2 micrometers, making it suitable for a range of filtration applications.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (23 mentions) Chemidoc mp imaging system, by Bio-Rad (9 mentions) Odyssey ir imaging system, by LI COR (8 mentions) Bovine serum albumin (bsa), by Merck Group (7 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (7 mentions) Ripa buffer, by Thermo Fisher Scientific (6 mentions) Amersham hyperfilm ecl, by GE Healthcare (5 mentions) Chemidoc imaging system, by Bio-Rad (5 mentions) Clarity western ecl substrate, by Bio-Rad (5 mentions) Image lab, by Bio-Rad (4 mentions) Trans blot turbo transfer system, by Bio-Rad (4 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (4 mentions) Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Odyssey blocking buffer, by LI COR (3 mentions) Ecl reagent, by Thermo Fisher Scientific (3 mentions) Fastprep 24 lysing matrix tubes, by MP Biomedicals (3 mentions) Phosphatase inhibitor, by Merck Group (3 mentions) Odyssey clx imaging system, by LI COR (3 mentions) Trans blot turbo system, by Bio-Rad (3 mentions)

Close spelling variants of this product

Nitrocellulose membranes (0.2 μm, Trans-Blot Turbo Mini 0.2 μm nitrocellulose membranes 0.2 μm nitrocellulose Nitrocellulose membranes Nitrocellulose

154 protocols using 0.2 μm nitrocellulose membrane

1

Western Blot Analysis of Lung Tissue

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Western blot analysis was performed as previously4 (link). In brief, lung tissue was collected and protein prepared in FastPrep-24 lysing matrix tubes (MP Biomedicals) then lysed using Radio-Immunoprecipitation Assay (RIPA) buffer (50 mM TricCl pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) containing protease (Calbiochem, San Diego, CA, USA) and phosphatase inhibitors (Sigma-Aldrich Corp.). 20 μg protein was run on an SDS 8–12% Tris-Glycine gel (Novex, Life Technologies) and then transferred onto a 0.2-μm nitrocellulose membrane (BioRad) overnight, blocked with 1% milk and probed for GR (1:1,000, clone M-20, sc-1004, Santa Cruz), Cav1 (1:1000, clone N20, Santa Cruz), Cavin (1:1000), actin (1:1000), TFIIB (1:1000, Santa Cruz) used to confirm nuclear lysis, Histone 3 (1:1000, Cell Signalling). Immunoreactivity was visualized using enhanced chemiluminescence (GE Healthcare). Densitometry was performed using ImageJ.
Caratti G., Poolman T., Hurst R.J., Ince L., Knight A., Krakowiak K., Durrington H.J., Gibbs J., Else K.J., Matthews L.C, & Ray D.W. (2019). Caveolin1 interacts with the glucocorticoid receptor in the lung but is dispensable for its anti-inflammatory actions in lung inflammation and Trichuris Muris infection. Scientific Reports, 9, 8581.
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2

SDS-PAGE and Western Blot Analysis

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BALF and proteins were resuspended in denaturing SDS sample buffer and resolved by SDS-PAGE and immunoblotted as described earlier [20 (link)]. Briefly, samples were resolved on 4–15% SDS PAGE, transferred onto 0.2 μm nitrocellulose membrane (BIORAD, Hercules, CA), probed with primary antibodies, washed, then incubated with secondary HRP-conjugated antibodies or avidin-HRP. Western blot detection was performed using ECL plus (Amersham, Arlington Heights, IL).
Ménoret A., Svedova J., Behl B, & Vella A.T. (2015). Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation. PLoS ONE, 10(10), e0141548.
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3

Western Blot Analysis of DECR in Mouse Tissues

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Because of small sample sizes, protein samples of iBATs from mice of each treatment and genotype groups (wild type and Decr−/−) were pooled for Western blot analysis. One pooled sample composed of equal amounts of protein from 5 mice adding up to 50 μg of protein, which was loaded per well in SDS-PAGE and transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) using a Trans-Blot Turbo-system (Bio-Rad). The membranes were blocked using 5% BSA in TBS 0.1% Tween. Primary antibodies and horseradish peroxidase conjugated secondary antibodies were used to detect proteins of interest (Supplementary Table S1). To analyze the amount of DECR in mouse iBAT and WAT, 15 µg of protein sample from individual mouse iBAT and 30 µg of protein sample from individual mouse WAT were used for a blot and a polyclonal antibody raised against rat 2,4-dienoyl-CoA reductase6 (link) was used as a primary antibody. Antibody detection reaction was developed using an Immun-StarTM WesternCTM Chemiluminescent Kit (Bio-Rad), Molecular Imager ChemiDocTM XRS+ equipment and Image Lab version 3.0 software (Bio-Rad). β-Actin was used as a loading control and for quantification of detected proteins with Image Lab software.
Mäkelä A.M., Hohtola E., Miinalainen I.J., Autio J.A., Schmitz W., Niemi K.J., Hiltunen J.K, & Autio K.J. (2019). Mitochondrial 2,4-dienoyl-CoA reductase (Decr) deficiency and impairment of thermogenesis in mouse brown adipose tissue. Scientific Reports, 9, 12038.
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4

Dot Blot Analysis of LKSs

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LSKs were sorted, irradiated and cultured in F12 complete media for 1 hour. Then, cells were lysed in modified Laemmli buffer (240 mM Tris/HCl pH 6.8, 8% SDS, 5% beta-mercaptoethanol) at 5×105 cells/ml and incubated 10 minutes at 99° C. 30×105 LKSs were spotted on a 0.2μm nitrocellulose membrane (BioRad), let dry, washed with PBST (0.1% Tween20 in PBS) and blocked overnight at 4° C in 5% BSA in PBST. Primary antibodies (anti -H2A.X Phospho Ser139 from BioLegend and actin from Santa-Cruz) were used at 1/1000 for 2 hours RT in 0.5% BSA in PBST. Secondary antibodies (anti-mouse HRP, Santa Cruz and anti-goat HRP, Jackson) were incubated at 1/2000 in 0.5% BSA in PBST. Membranes were washed 3 times for 15 minutes in PBST. Dot blot was developed using SuperSignal West Femto (ThermoFisher Scientific) and Amersham Hyperfilm ECL (GE Healthcare).
Gutierrez-Martinez P., Hogdal L., Nagai M., Kruta M., Singh R., Sarosiek K., Nussenzweig A., Beerman I., Letai A, & Rossi D.J. (2018). Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage. Nature cell biology, 20(4), 413-421.
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5

Quantitative Analysis of Signaling Proteins

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Western Blots were performed with 20 μg protein from each sample. After 48 hours transfection, WAC3CD5+ cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% SDS, 1% Triton X-100, 2 mM EDTA). Protein lysates were separated on 10% SDS-PAGE and blotted onto a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules, CA). The primary antibodies used were for anti-MADD (1:2000; Abcam), -PIK3R2 (1:1000; Abcam), -phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000; Cell Signaling), -p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling), -phospho-Akt (Thr308) (1:2000; Cell Signaling), -phospho-AKT (Ser473) (1:2000; Cell Signaling), -AKT (1:1000; Cell Signaling) and anti-actin (1:5000; Sigma-Aldrich, USA) at 4°C overnight. Then membranes were washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase conjugate secondary antibody at room temperature for 1 hour. ECL plus Western blotting detection reagent was used for the detection of protein signals with X-ray film (Amersham Biosciences, Buckinghamshire, UK). Protein bands were quantified using densitometry as measured by Quantity One 4.6.2 software (Bio-Rad); hence relative protein expression was expressed in comparison to corresponding control.
Wang L.Q., Wong K.Y., Rosèn A, & Chim C.S. (2015). Epigenetic silencing of tumor suppressor miR-3151 contributes to Chinese chronic lymphocytic leukemia by constitutive activation of MADD/ERK and PIK3R2/AKT signaling pathways. Oncotarget, 6(42), 44422-44436.
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6

Western Blot Quantification Protocol

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Following treatments, cells were washed with cold PBS and lysed in 1x Laemmli buffer. The protein concentrations in the cell lysates were measured using Pierce BCA protein assay Kit (ThermoFisher, #23227). The same amount protein for each sample (20-50 µg/lane) was loaded to SDS-PAGE gel, followed by transferring proteins to a 0.2μm nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked with blocking buffer Blocker Casein in PBS (ThermoFisher, #37582) at room temperature for 1 h, followed by immunoblotting with the primary antibodies in 5% BSA TBST buffer at 1:1000 dilution at room temperature overnight. After washing with TBST buffer, the immunoblotting membrane was then probed with the secondary antibodies with fluorescence conjugation at 1:10000 dilution. The immunoblots were visualized and the immunoblotting signal intensity quantitated using the Odyssey IR imaging system (Li-Cor Biosciences).
Zhao M., DiPeri T.P., Raso M.G., Zheng X., Rizvi Y.Q., Evans K.W., Yang F., Akcakanat A., Roberto Estecio M., Tripathy D., Dumbrava E.E., Damodaran S, & Meric-Bernstam F. (2023). Epigenetically upregulating TROP2 and SLFN11 enhances therapeutic efficacy of TROP2 antibody drug conjugate sacitizumab govitecan. NPJ Breast Cancer, 9, 66.
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7

Western Blot Analysis of Neuronal Proteins

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Human samples were homogenized in RIPA lysis buffer with proteinase inhibitors (Santa Cruz Biotechnology, Dallas, TX, United States, sc24948). Protein extracts, 40 μg per lane, were loaded onto 4–20% gradient gels (NuSep Inc., Germantown, MD, United States, NB10-420). Gels were electrotransferred to a 0.2 μm nitrocellulose membrane (Bio-Rad, 1620174). Blots were blocked in 5% milk in tris-buffered saline, 0.1% Tween 20 (TBST) for 1 h, and then incubated at 4 °C overnight with one of the following antibodies raised against: AATM (1:1000, sc-271702, Santa Cruz Biotechnology), AQP4 (1:1000, sc-390488), CX30 (1:1000, sc-81802), CX43 (1:1000, sc-271837), EAAT1 (1:500, sc-515839), GABRA1-6 (1:250, sc-376282), GAPDH (1:2000, sc-32233), GAT-3 (1:250, sc-376001), GDH1/2 (1:1000, sc-515542), GEPH (1:1000, sc-25311), GS (1:1000, sc-74430), MAOB (1:250, sc-515354), SNAT5 (1:1000, sc-515813), VIAAT (1:500, sc-393373), GBRD (1:250, Novus Biologicals, Littleton, CO, United States, NB300-200) or GABRG1 (1:100, Alomone Labs, Jerusalem, Israel, AGA-016). Bands were detected with appropriate horseradish peroxide-conjugated secondary antibodies, reacted with chemiluminescent ECL substrate (Bio-Rad, 1705060) and visualized with a Bio-Rad ChemiDoc Imaging system. Band intensity was measured using the ImageJ program (NIH) and normalized with GAPDH.
Lacaille H., Vacher C.M, & Penn A.A. (2022). Preterm Birth Alters the Maturation of the GABAergic System in the Human Prefrontal Cortex. Frontiers in Molecular Neuroscience, 14, 827370.
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8

Comparative Western Blot Analysis of Protein Extracts

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Protein extracts of 107 of either uRBCs or iRBCs and of the breast cancer cell line MDA-MB 231 (used as a positive control and kindly provided by Dr. Delphine Merino, Olivia Newton-John Cancer Research Centre) were loaded on a 4–12% Bis-Tris gel (NovexTM) and resolved in MES-SDS running buffer (NovexTM). Proteins were transferred onto a 0.2 μm nitrocellulose membrane (BioRad, Hercules, CA, USA) in transfer buffer (0.3% Tris base, 0.75% glycine, 20% methanol), and the membrane was washed in PBST (0.1% Tween-20 in PBS) for 10 min and then blocked overnight at 4 °C in 5% milk-PBST. Primary and secondary antibodies were incubated successively, overnight at 4 °C and for 1 h at room temperature, respectively. Dilutions of primary antibodies (in 5% BSA-PBST) were used as follows: BCL-xL 1:1000 (Cell Signaling, Danvers, MA, USA, #2764), Carbonic Anhydrase 1:4000 (Abcam, Cambridge, UK, #ab108367), PfHSP70.1 1:5000 (kindly provided by Prof Gilson and Prof Crabb, Burnet Institute), and protein 4.1 1:500 (kindly provided by Dr. Proellocks and Prof Cooke, Monash University). Secondary antibody: anti-rabbit-HRP 1:5000 (Cell Signaling, #7074). HRP signal was revealed with Pierce™ ECL Western Blotting Substrate (Thermo Scientific) and detected with a ChemiDoc XRS+ system.
Boulet C., Siddiqui G., Gaynor T.L., Doerig C., Creek D.J, & Carvalho T.G. (2022). Red Blood Cell BCL-xL Is Required for Plasmodium falciparum Survival: Insights into Host-Directed Malaria Therapies. Microorganisms, 10(4), 824.
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9

Western Blot Analysis of Protein Targets

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Cell lysates were diluted with 4x Laemmli Sample buffer (Biorad) and run on 4–20% SDS-PAGE denaturing gel (Biorad Cat#4561096) under reducing conditions (180V for 40 minutes). Protein was transferred to 0.2 μm nitrocellulose membrane (Bio-Rad) using the Trans-Blot Turbo semi-dry transfer apparatus. Blots were blocked in 5% milk in PBST and primary antibodies were diluted into this solution for staining. Primary polyclonal antibodies were purchased from the following suppliers and used at the indicated dilutions: CYB5B (RRID AB_2230349, 1:1000), C18orf32 (RRID AB_2638853, 1:500), Rpn13/ADRM1 (RRID: AB_2225663, 1:1000), REEP5 (RRID AB_2178440, 1:1000), OCIAD1 (RRID AB_11035850, 1:1000), GAPDH (RRID: AB_627679, 1:1000), FLAG (RRID: AB_262044, 1:1000),β5 proteasome subunit (RRID: AB_2052392, 1:1000), Uch37 (RRID: AB_2814821, 1:1000). Primary antibodies were incubated with membrane overnight at 4°C. Secondary antibodies IRDye 680RD donkey anti-mouse IgG (RRID: AB_27116622) and IRDye 800CW goat anti-rabbit IgG (RRID: AB_2651127) were used at 1:10,000 dilution with a 1 hour incubation at room temperature. Streptavidin IR680 RD (Licor #926–68079) was used at a 1:5000 dilution to detect biotin-RA190 protein adducts. Secondary antibody was diluted into Licor Oddysey blocking buffer. Blots were resolved using Licor Oddysey and Licor Image Studio Software (RRID: SCR_015795)
Dickson P., Abegg D., Vinogradova E., Takaya J., An H., Simanski S., Cravatt B.F., Adibekian A, & Kodadek T. (2020). Physical and Functional Analysis of the Putative Rpn13 Inhibitor RA190. Cell chemical biology, 27(11), 1371-1382.e6.
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10

Immunoblotting of Ubiquitin Pathway Proteins

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The proteins (E1, E2s, ubiquitin) were run on 12% Bolt Bis-Tris Plus SDS-PAGE gels (Thermo Fisher Scientific), transferred to a 0.2-μm nitrocellulose membrane (BioRad), and incubated with mouse anti-6His-tag antibody (1:500, Roche, cat. no 11 922 416 001), mouse anti-Ub antibody (1:1000, Enzo, cat. no ADI-SPA-203) or rabbit anti-Ube1 antibody (1:1000, Enzo, cat. no BML-PW8395), and infrared dye-coupled goat anti-mouse or anti-rabbit secondary antibody (1:20,000, LI-COR, cat. no 925-32210 and 925-32211). Blots were scanned using an infrared imager (Odyssey, LI-COR Biosciences) at 800 nm.
Koszela J., Pham N.T., Evans D., Mann S., Perez-Pi I., Shave S., Ceccarelli D.F., Sicheri F., Tyers M, & Auer M. (2018). Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay. BMC Biology, 16, 88.
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